A more rapid version of this protocol has been developed and is available here. It has been show to result in usable DNA from stool samples also.
DNA isolated from sources rich in carbohydrates is often refractory to manipulation by standard molecular biology techniques. Plan biologists and bacteriologists independently used cetyltrimethylammonium bromide (CTAB) to selectively remove these carbohydrates from their samples. This reagent has also proven useful in the isolation of DNA from Entamoeba using the method below. Entamoeba is known to store glycogen and standard DNA isolation methods often yield DNA that is difficult to digest with restriction enzymes or use as a template for polymerase chain reaction. Whether this is due to the glycogen is not established but it is quite likely to be the case. The same method has also been used to isolate DNA from a wide range of unicellular eukaryotes with excellent results.
My own experience has been only with material from in vitro cultures so that readers may want to find methods for DNA isolation directly from stool or other sources elsewhere. However, I know from work by other researchers that this method has been used successfully to isolate Entamoeba DNA from stool that was a suitable template for Polymerase Chain Reaction amplification.
Sample preparation from cultures
Xenic or axenic material from mono- or di-phasic medium is harvested by chilling (to release cells from the culture vessel) and centrifugation, followed by transfer of the pellet to a microcentrifuge tube, repelleting, and dispersal in 250 µl of lysis buffer (0.25% SDS in 0.1M EDTA pH 8.0). Samples are stable for months in this condition at 4° C and I have successfully shipped such lysates by mail.
1. A pellet of at least 50 µl packed volume can be processed with the following protocol. To the ca. 300 µl lysate, add Proteinase K to 100 µg/ml and incubate at 55° C for 1 hour.
2. Add 75 µl of 3.5 M NaCl, mix gently then add 42 µl of 10% CTAB/0.7 M NaCl (Note) (heated to 55° C), mix and incubate at 65° C for 20 minutes.
3. At room temperature add 400 µl of chloroform, mix well by inverting and spin at full speed in a microcentrifuge for 10 minutes.(Note)
4. Transfer the supernatant to a fresh tube and add 400 µl of phenol:chloroform:isoamyl alcohol (25:24:1), mix well by inversion and spin as above.
5. Transfer the supernatant to a fresh tube and add two volumes of 100% ethanol, mix by inversion, store at room temperature for at least 5 minutes and then spin 15 minutes as above.
6. Carefully discard the supernatant from the pellet and wash the pellet in 200 µl of 70% ethanol by spinning for 5 minutes as above.
7. Air dry the pellet and resuspend it in up to 50 µl water.
8. Pass the resuspended DNA over a Sephacryl S400 spin column (0.5 ml packed matrix; 850 x g 5 minutes). (Note)
Note 1: CTAB is a cationic detergent that binds polysaccharides under these salt conditions. It is important to maintain the NaCl concentration above 0.5 M or a CTAB-DNA precipitate will form. Heating at 65° C will be necessary to dissolve the 10% CTAB and the stock should be reheated each time before use to reduce viscosity. (Back)
Note 2: This step precipitates the CTAB-Polysaccharide complex. The interface can be very large and the recovered volume much less than expected, but in my experience reextracting the interface with 0.7 M NaCl yields little additional DNA. (Back)
Note 3: Although I have had success using DNA directly after resuspension, the Sephacryl S400 can remove certain compounds that are inhibitory to PCR, restriction enzymes, etc. without loss of DNA. DNA can be stored at -20° C. (Back)
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